Cancer is the major cause of mortality in most industrialized countries. Different ways of cancer treatment can be used: surgery, radiotherapy, immunotherapy, hormonotherapy and chemotherapy. Numerous research laboratories lead works to find cancer therapy improvements. Chemotherapy leads to the cell death. Two type of cell death are known: the apoptosis and the necrosis.
It has long been hypothesized that apoptotic cell death would be poorly immunogenic (or even tolerogenic) whereas necrotic cell death would be truly immunogenic (Bellamy, C. O., Malcomson, R. D., Harrison, D. J. & Wyllie, A. H. Semin Cancer Biol 6, 3-16 (1995); Thompson, C. B. Science 267, 1456-1462 (1995); Igney, F. H. & Krammer, P. H. Nat Rev Cancer. 2, 277-88 (2002)).
This difference was thought to result from the intrinsic capacity of cells dying from non-apoptotic cell death to stimulate the immune response, for example by stimulating local inflammatory responses (‘danger signals’) and/or by triggering the maturation of dendritic cells (DCs) (Steinman, R. M., Turley, S., Mellman, I. & Inaba, K. J Exp Med. 191, 411-6 (2000); Liu, K. et al. J Exp Med 196, 1091-1097. (2002).
In contrast to necrosis (which is defined by brisk plasma membrane rupture), apoptosis is associated with a series of subtle alterations in the plasma membrane that render the dying cells palatable to phagocytic cells (Kroemer, G. et al. Cell Death Differ 12, 1463-1467 (2005)). Such “eat me” signals, which include the adsorption of soluble proteins from outside the cell (such as C1q and thrombospondin) and the translocation of molecules from inside the cell to the surface (such as phosphatidylserine, PS, and calreticulin, CRT), as well as the suppression of “don't eat me” signals (such as CD47) (Savill, J. & Fadok, V. Nature 407, 784-(2000); Lauber, K., Blumenthal, S. G., Waibel, M. & Wesselborg, S. Mol Cell. 14, 277-87 (2004); Yoshida, H. et al. Nature 437, 754-8 (2005); Gardai, S. J. et al. Cell 123, 321-34 (2005); Henson, P. M. & Hume, D. A. Trends Immunol 27, 244-50 (2006)) elicit the recognition and removal of apoptotic cells by professional and non-professional phagocytes. Suboptimal clearance of apoptotic cells can trigger unwarranted immune reactions and lead to autoimmune disease (Hanayama, R. et al. Science 3004, 1147-50. (2004); Gaipl, U. S. et al. Curr Top Microbiol Immunol 305, 161-76 (2006)).
Nonetheless, it seems that the dichotomy between immunogenic necrosis versus tolerogenic apoptosis is an oversimplification. Thus, unscheduled (necrotic) tumour cell death may induce local immunosuppression (Vakkila, J. & Lotze, M. T Nat Rev Immunol 4, 641-8 (2004)). Moreover, the capacity of apoptotic tumour cells to trigger the immune response was found to depend on the apoptosis inducer, leading to the identification of two morphologically undistinguishable subcategories of apoptosis, namely immunogenic versus non-immunogenic apoptosis (Casares, N. et al. J. Exp. Med. 202, 1691-701 (2005); Blachere, N. E., Darnell, R. B. & Albert, M. L. PLoS Biol. 3, e185 (2005)).
Most of standard chemotherapies induce a non-immunogenic apoptosis (Zitvogel, L., Casares, N., Pequignot, M., Albert, M. L. & Kroemer, G. Adv. Immunol. 84, 131-79 (2004); Steinman, R. M. & Mellman, I. Science 305, 197-200 (2004); Lake, R. A. & van der Most, R. G. N Engl J Med 354, 2503-4 (2006)). Thus, even after an initially efficient chemotherapy, patients do not develop an efficient antitumourous immune response and then are overcome by chemotherapy-resistant tumourous variants. To improve anticancer chemotherapy, a promising way is brought by the immunogenic cancer-cell death. Indeed, induction of immunogenic cancer-cell death should be very interesting in that the immune system can contribute through a “bystander effect” to eradicate chemotherapy-resistant cancer cells and cancer stem cells (Steinman, R. M. & Mellman, I. Science 305, 197-200 (2004); Lake, R. A. & van der Most, R. G. N Engl J Med 354, 2503-4 (2006); Zitvogel, L., Tesniere, A. & Kroemer, G. Nat Rev Immunol in press (2006)).
The efficiency of chemotherapy and the responsiveness is linked to the drugs used and to the molecules involved in the chemotherapy. The main drugs used in anti-tumourous chemotherapy can be divided in four groups: cytotoxic agents, hormones, immune response modulators, and inhibitors of the tyrosine kinase activity. Among cytotoxic agents, one can find cytotoxic antibiotics such as anthracyclins (doxorubicin, idarubicin, mitoxantrone which are apoptosis inducers). Inventors have shown for the first time that anthracyclins are capable of eliciting immunogenic apoptosis (Casares, N., Pequignot, M. O., Tesniere, A., Ghiringhelli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A., Hervas-Stubbs, S., Obeid, M., Coutant, F., Métivier, D., Pichard, E., Aucouturier, P., Pierron, G., Garrido, C., Zitvogel, L., and Kroemer, G. J Exp Med. 202, 1691-701 (2005)). Indeed, while most apoptosis inducers, including agents that target the endoplasmic reticulum (ER) (thapsigargin, tunicamycin, brefeldin), mitochondria (arsenite, betulinic acid, C2 ceramide) or DNA (Hoechst 33343, camptothecin, etoposide, mitomycin C), failed to induce immunogenic apoptosis, anthracyclins elicited immunogenic cell death (as shown in FIGS. 1B,C). Despite a growing body of research, under which circumstances an immune response is triggered against dying tumour cells remains an open question (Zitvogel, L., Casares, N., Pequignot, M., Albert, M. L. & Kroemer, G. Adv. Immunol. 84, 131-79 (2004)).
The present invention is based on the observation that the proteins named calreticulin (CRT), KDEL receptor (KDEL receptor is a CRT receptor) and ERp57 are exposed on cells that succumb to immunogenic cell death, yet lacks on the surface of cells that undergo non-immunogenic cell death.
CRT has been already described for modulating the hormonal response, another way to treat cancer. Proteins which modulate hormone receptor induced gene transcription are present in the nucleus of the cell and inhibit or promote the binding of a hormone to its receptor. The invention described in the patent US 2003/0060613 A1 presents a purified protein, efficient in anti-cancer therapy, which is used to modulate hormone responsiveness. US 2003/0060613 A1 describes CRT as a synthetic protein, a mimetic protein thereof, a DNA molecule encoding CRT, a method of treating a disease such as cancer, and a kit containing a pharmaceutical comprising one of said proteins. CRT is described to be present either in the endoplasmic reticulum or in the nucleus of a cell. This patent application describes mechanism of action of nuclear CRT on gene transcription.
ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 is known to interact specifically with newly synthesized glycoproteins including CRT. Oliver et al. (MBC online, Vol. 10, Issue 8, 2573-2582, August 1999) indicates that ERp57 interacts with calnexin and CRT within the ER lumen to modulate glycoprotein folding.
The majority of endoplasmic reticulum resident proteins are retained in the endoplasmic reticulum (ER) through a retention motif. This motif is composed of four amino acids at the end of the protein sequence. The most common retention sequence is KDEL (Lys-Asp-Glu-Leu) (SEQ ID NO:16). There are three KDEL receptors in mammalian cells, and they have a very high degree of sequence identity. The KDEL receptor (KDEL-R) is usually present within the ER. Inventors have shown that KDEL receptor can also interact with the KDEL motif of CRT.
The inventors have more particularly identified a particular alteration in the plasma membrane of dying cells: the surface exposure of calreticulin (CRT), KDEL receptor and/or ERp57, This event only occurs in immunogenic cancer cell death, i.e., in cancer cell death implying a reaction of the immune system. In immunogenic cancer cell death, the immune system thus participates to the dying cells elimination in addition to the other physiological cell destruction mechanisms. Exogenous CRT, KDEL receptor and/or ERp57, or external provision of signals that induces CRT, KDEL receptor and/or ERp57 exposure, confers immunogenicity to otherwise non-immunogenic cell death, i.e., mobilize and activate the immune system, to allow an optimal anti-cancer chemotherapy.